Summary: This grant application continues my previous work, focusing on integrating stem cell biology, cancer stem cells and immunology to understand: i) mechanisms controlling stem cell numbers, self-renewal and differentiation; ii) the genetic/epigenetic `events' that drive cancer initiation and progression through clonal expansion and competition of stem cells; iii) mechanisms of programmed cell removal of precancerous cells by macrophage phagocytosis and how cancer cells evade those, and iv) developing innate system [mainly macrophage-based] cancer immunotherapies. I was awarded an NCI OIG 28 years ago and used the funding to develop the first method to identify and isolate blood forming stem cells [HSC]. We applied this discovery in a clinical trial where autologous transplantation of purified, cancer-free HSC from mobilized peripheral blood [MPB] to metastatic breast cancer patients following high dose chemotherapy, resulted in 33% 18-20 year survival compared to 7% with MPB. We then mapped the differentiation steps from stem cells to all mature blood cells and in the context of myeloid leukemogenesis, studied how genetic alterations modify the affected stem cells. We isolated leukemic stem cells[LSC] that could regenerate myelogenous leukemia and found that these cells were not phenotypic HSC but downstream MPP progenitors. Although many HSC in the same patients had the cancer-initiating mutations, they were not leukemic! Additional mutations or `events' were required, each promoting clonal expansion and competition of the preleukemic HSC over normal HSC. This model of sequential progression of events accruing one at a time in HSC `clones' until the LSC emerges holds true for all myeloid leukemias and preleukemias. Comparing LSC to HSC we discovered that the expression of CD47, a `don't eat me' signal for macrophage scavenger cells, was a late event for all cancers including leukemias. We made blocking antibodies to CD47, and found that these led to tumor cell phagocytosis and cancer regression and often cures in xenograft models of human leukemias and solid tumors. Anti-CD47 antibody synergizes with all other anti- cancer antibodies tested to date. The current proposal expands on these studies to test whether accumulation of mutations also occurs in a central nervous system stem cell(CNS SC) clone that gives rise to a brain cancer stem cell [CSC]. The gene expression profiles of cells from these precancers and cancers should identify new therapeutic targets. Importantly, normal C47+ cells aren't eaten when CD47 is blocked, because they lack a pro-phagocytic `eat me signal', calreticulin, that is on all cancers. We will study how cancer cells are labeled with calreticulin for elimination by macrophages, and extend our macrophage phagocytosis studies to try to understand how macrophages get rid of old, damaged, and dying cells, and how pathogenic stem cells avoid being eaten via CD47 or other `don't eat me' signals. In these studies we will examine additional stem cell systems that when gone awry lead to cancer and other diseases.